Multi-Level Biological Responses in Ucides Cordatus

Ecotoxicology and Environmental Safety 133 (2016) 176–187

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Multi-level biological responses in Ucides cordatus (Linnaeus, 1763) (Brachyura, Ucididae) as indicators of conservation status in mangrove areas from the western atlantic

Luis Felipe de Almeida Duarte a,n, Caroline Araújo de Souza a, Caio Rodrigues Nobre b, Camilo Dias Seabra Pereira c, Marcelo Antonio Amaro Pinheiro a a UNESP - Univ Estadual Paulista, Instituto de Biociências (IB), Campus do Litoral Paulista (CLP), Laboratório de Biologia de Crustáceos/CRUSTA, Praça Infante Dom Henrique, s/n, Par - Grupo de Pesquisa em Biologia Laboratório de Biologia de Crustáceos-Praça, Infante D. Henrique, Campus Experimental do Litoral Paulista (CLP), s/n, Parque Bitaru, 11330-900 São Vicente, São Paulo, Brazil b UNISANTA - Univ Santa Cecília, Laboratório de Ecotoxicologia, Oswaldo Cruz 266, 11045-907 Santos, SP, Brazil c UNIFESP - Univ Federal de São Paulo, Departamento de Ciências do Mar, Campus Baixada Santista, Avenida Almirante Saldanha da Gama 89, Ponta da Praia, 11030400 Santos, SP, Brasil article info abstract

Article history: There is a global lack of knowledge on tropical ecotoxicology, particularly in terms of mangrove areas. Received 19 April 2016 These areas often serve as nurseries or homes for several animal species, including Ucides cordatus (the Received in revised form uçá crab). This species is widely distributed, is part of the diet of human coastal communities, and is 12 July 2016 considered to be a sentinel species due to its sensitivity to toxic xenobiotics in natural environments. Accepted 13 July 2016 Sublethal damages to benthic populations reveal pre-pathological conditions, but discussions of the Available online 22 July 2016 implications are scarce in the literature. In Brazil, the state of São Paulo offers an interesting scenario for Keywords: ecotoxicology and population studies: it is easy to distinguish between mangroves that are well pre- Environmental safety served and those which are significantly impacted by human activity. The objectives of this study were to Cytotoxicity provide the normal baseline values for the frequency of Micronucleated cells (MN‰) and for neutral red Density retention time (NRRT) in U. cordatus at pristine locations, as well to indicate the conservation status of Genotoxicity Mangrove different mangrove areas using a multi-level biological response approach in which these biomarkers Ucides cordatus and population indicators (condition factor and crab density) are applied in relation to environmental quality indicators (determined via information in the literature and solid waste volume). A mangrove area with no effects of impact (areas of reference or pristine areas) presented a mean value of MN‰o3 and NRRT4120 min, values which were assumed as baseline values representing genetic and physiological normality. A significant correlation was found between NRRT and MN, with both showing similar and effective results for distinguishing between different mangrove areas according to conservation status. Furthermore, crab density was lower in more impacted mangrove areas, a finding which also reflects the effects of sublethal damage; this finding was not determined by condition factor measure- ments. Multi-level biological responses were able to reflect the conservation status of the mangrove areas studied using information on guideline values of MN‰, NRRT, and density of the uçá crab in order to categorize three levels of human impacts in mangrove areas: PNI (probable null impact); PLI (probable low impact); and PHI (probable high impact). Results confirm the success of U. cordatus species’ multi- level biological responses in diagnosing threats to mangrove areas. Therefore, this species represents an effective tool in studies on mangrove conservation statuses in the Western Atlantic. & 2016 Elsevier Inc. All rights reserved.

1. Introduction biodiversity and generate approximately 60% of the primary productivity of the planet, ecotoxicological studies have been con- Although tropical ecosystems house 75% of the world’s ducted almost exclusively in temperate ecosystems (Peters et al., 1997; Lacher and Goldstein, 1997; Sueitt et al., 2015). Such limited knowledge contrasts with current increasing human impacts in n Corresponding author. the tropics, impacts which are generally the result of unsustain- E-mail addresses: [email protected] (L.F.d.A. Duarte), [email protected] (C.A.d. Souza), [email protected] (C.R. Nobre), able activities and the release of xenobiotics into water bodies. [email protected] (C.D.S. Pereira), [email protected] (M.A.A. Pinheiro). These activities ultimately have negative effects on the aquatic http://dx.doi.org/10.1016/j.ecoenv.2016.07.018 0147-6513/& 2016 Elsevier Inc. All rights reserved.

Please cite this article as: Duarte, L.F.d.A., et al., Multi-level biological responses in Ucides cordatus (Linnaeus, 1763) (Brachyura, Ucididae) as indicators of conservation status in mangrove areas from the western atlantic. Ecotoxicol. Environ. Saf. (2016), http://dx.doi. org/10.1016/j.ecoenv.2016.07.018i L.F.d.A. Duarte et al. / Ecotoxicology and Environmental Safety 133 (2016) 176–187 177 biota (Peters et al., 1997; Harford et al., 2015). between this bioaccumulation and the genetic impacts observed Different approaches, which consider information ranging from (Pinheiro et al., 2012, 2013). As a result, these xenobiotics are data at the population-community level to sentinel species’ sub- magnified by the trophic chain of the mangrove (Fiscarelli and lethal responses, are the current basis for most biomonitoring Pinheiro, 2002). This species also has a close relationship with programs (Marine Strategy Framework Directive EU - MSFD, 2008/ three environmental compartments of mangroves with which it 56/EC). Short-term responses among lower levels of biological interacts and where it absorbs pollutants: (1) water, via contact, organization have been used to detect the first signs of impair- ingestion, the respiration process, and metabolite excretion; ment caused by xenobiotics (Amiard-Triquet et al., 2013; Pereira (2) sediment, via contact when digging burrows and mud feeding; et al., 2014). These responses have generally been referred to as and (3) trees, through their feeding on the senescent leaves and “biomarkers,” which are defined herein as genetic, biochemical, seedlings (Fiscarelli and Pinheiro, 2002; Pinheiro et al., 2013; cellular, physiological, or behavioral variations that can be mea- Christofoletti et al., 2013). sured in tissue or body fluid samples or at the level of the whole A multi-level biological response approach must include not organism and which provide evidence of exposure to and/or ef- only the most ideal biological model – the area selected for study fects of one or more chemical pollutants and/or radiations (De- must also present a wide regional environmental impact gradient pledge et al., 1993). so that the investigation will be more effective and meaningful. Genetic and physiological changes are therefore of particular Therefore, it is important to highlight the high anthropic impact importance, since they are conspicuous in organisms that inhabit observed in southeastern Brazil, particularly in coastal areas. impacted areas (Adams, 1990; Dailianis et al., 2003; Otomo and However, pristine mangroves also exist and are represented by Reinecke, 2010; Amiard-Triquet et al., 2013; Toufexia et al., 2013). protected areas (Cetesb, 2001, 2007). It is, therefore, a contrasting Because of their effectiveness and ecological relevance (Bonassi scenario that has been mentioned and studied by several authors et al., 2000; Dailianis et al., 2003; Neri et al., 2003), two biomarker (Silva et al., 2002; Abessa and Ambrozevicius, 2008; Cordeiro and assays are widely used. The first is the micronucleus test (MN‰), Costa, 2010; Martins et al., 2011; Pereira et al., 2011; Torres et al., which quantifies the frequency of micronucleated cells and which 2012; Pinheiro et al., 2008, 2012, 2013). measures genotoxicity. Genotoxicity is higher in more highly im- Given both the contamination gradient of the mangroves in the pacted areas (Brunetti et al., 1988; Burgeot et al., 1995; Collier region and the possible biological responses at the cellular, phy- et al., 2013; Pinheiro et al., 2013). The second is the neutral red siological, and population levels of a sentinel species (U. cordatus), retention time (NRRT) assay, which evaluates lysosomal mem- this study aims to: (1) provide normal baseline values for the brane stability as a diagnostic biomarker of individual health sta- micronucleated cell frequency (MN ‰) and neutral red retention tus (Buratti et al., 2012, Ospar, 2013). time (NRRT) in U. cordatus at pristine locations; and (2) perform a Genetic and physiological changes are considered pre-patho- multi-level assessment using this species as a biological model by logical indicators (Tsarpali and Dailianis, 2012) that can detect analyzing the effectiveness and ecological relevance of two bio- biological dysfunction in response to a stressor before population markers (genetic via MN ‰ and physiological via NRRT), their problems occur (Amiard-Triquet et al., 2013). As determined by correlation with population parameters (condition factor and Adams, (1990) and by Fishelson et al. (1999), such sublethal da- density), and their interactions with the conservation status of mages may have more serious effects, such as increases in disease, different mangrove areas (determined via information from the changes to mortality rates, and population decline. According to literature and solid waste volume). The validation of this multi- these authors, other parameters found at higher levels of biolo- level approach will allow for the conservation statuses of the gical organization, such as at the population level, may also be mangroves to be categorized without the need for contaminant relevant bioindicators of environmental impact and quality (Bru- quantification or qualification. High costs are therefore avoided, ner et al., 2001; Barrilli et al., 2015). Therefore, Fulton's condition which will provide more opportunities for diagnosing this eco- factor (K) would be a relevant population parameter in these as- system's conservation status. sessments: it can quantify “well-being” as a ratio of size to individual weight (Lima-Junior et al., 2002), with higher values in- dicating better environmental quality (Viana et al., 2014; Barrilli 2. Materials and methods et al., 2015). Similarly, density is a parameter that can be reduced by contaminants, as seen among crustaceans (Krebs and Valiela, 2.1. Mangrove areas studied and their conservation statuses 1974; Krebs and Burns, 1977), although the reverse effect can also occur, leading to an increase in density due the tolerance of con- The coast of São Paulo State in Brazil is frequently divided into taminants and their indirect effects on the environment (Cannicci three sections referred to as the northern coast, the central coast, et al., 2009). and the southern coast. These sections are distinguished by their There is limited knowledge available on links between sub- coastal plain areas (6000 km2, 3200 km2, and 7800 km2, respec- lethal damages seen in the lower levels of biological organization tively) (Gouveia, 2012), by the extent of river basins (194,800 ha, and those seen in higher levels (at the population or community 281,800 ha, and 1,706,800 ha, respectively; see BRASIL (2006)), level), particularly when a multi-level approach is considered. and by the extent of mangroves (central: 8858 ha; southern: However, in order for the biological model to be effective, it must 15,193 ha; see Cunha-Lignon et al. (2011a, 2011b) and Cunha-Lig- consider a species that is both important to the ecosystem and non (2014)). The present study considered mangroves from only sensitive enough to reveal early environmental disturbances the central coast (8858 ha) and the southern coast (15,193 ha), (Beltrame et al., 2010, 2011; Pereira et al., 2012, 2014). Thus, Pin- which represent the vast majority of the state’s mangrove area heiro et al. (2012) describe characteristics of the uçá crab (Ucides (99%). These sections also have a history of human occupation and cordatus) that reflect its importance as a sentinel species in man- impact dating back to the 1500 s when the first Brazilian city (São groves in the Western Atlantic: endemism and reduced mobility, Vicente) was founded on the central coast of the state (Cunha- bioturbation of sediments, feeding on leaves and sediments (de- Lignon et al., 2011b). Six mangrove areas have been established posit feeding); reduced growth, a relatively long lifespan, higher (Fig. 1), three of which are located on the central coast (Bertioga, or abundance, and increased ease of capture. The uçá crab accumu- BET, Cubatão, or CUB, and São Vicente, or SAV) and the other three lates contaminants in its tissues; there is a high correlation which are located on the southern coast (Iguape, or IGU; Cananéia,

Fig. 1. Location of the six mangrove areas sampled in the state of São Paulo (Brazil), represented by 18 subareas located in the central and southern coasts (Source: Satellite images from Google Earths redesigned by Gustavo Pinheiro). or CAN, and Juréia, or JUR, the latter of which is an ecological pentachlorophenol (PCP) into the environment. These three areas station). Each mangrove area includes three subareas (replicas) therefore have a history of more substantial human impact, with that are similar in tree composition (450% Rhizophora mangle), records of contamination by various xenobiotics, including metals vegetation structure (tree height 45 m and diameter at breast (Azevedo et al., 2009; Pinheiro et al., 2012, 2013) and organo- height 410 cm), height of flooding by tides (430 cm) and pro- chlorines (Martins et al., 2011). As a result, some sublethal damage ductivity (leaves available on the sediment 42gm2). However, has been observed in the local biota, including genetic changes the six areas have experienced different types of human occupa- reflected by an increase in micronucleus frequency and alterations tion and impacts, and different contamination levels. Each area has in the cell nucleus. These changes have been confirmed in fishes, been characterized according to relevant parameters (see Table 1) crabs, and mollusks (Kirschbaum et al., 2009; Caricato et al., 2010; and based on a review of the main pollutants recorded at con- Hagger et al., 2010; Dissanayake et al., 2011; Pinheiro et al., 2013). centrations above legally permitted levels for brackish water The mangrove areas on the southern coast of São Paulo are (Environment Canada, 1999a) and sediment (Environment Canada approximately 80–200 km away from the mangrove areas on the 1999b, FDEP 1994). central coast. The southern coast is comprised of estuaries with Located on the central coast of São Paulo, the Bertioga Estuary more conserved areas, though some of these areas have already (BET) receives domestic untreated sewage and effluents from a exhibited effects of indirect anthropogenic influence. The Estuar- public garbage dump as its main source of pollution. Even so, the ine System that includes Cananéia, Iguape, and Juréia is located estuary is used for ecotourism and sport fishing, including uçá crab within a single environmental protection area (EPA) that also in- fishing (Eichler et al., 2006). Cubatão (CUB) is considered one of cludes the entire state of Paraná. Together, these land masses form the most polluted regions in the world; in recent years, it has the Lagamar Complex, one of the largest estuarine complexes in become the most important Brazilian industrial hub, formed by 23 Brazil. It covers 3287 km2 and includes other interconnected ba- industrial complexes, 111 factories, and more than 300 pollution sins (ISA, 1998). Located in the northeastern most portion of this sources. Home to the largest port in Latin America, it also ex- complex is the area known as Iguape (IGU), which until the early periences large vessel transit in which several types of chemicals 1990s received significant waste discharge from rich heavy metal are imported and exported (Pinheiro et al., 2012, 2013). Located mining in the Ribeira River (approximately 5.5 metric tons per within the same estuarine complex is São Vicente (SAV). This area month of Pb, Zn, Ag, As, Cd, Cu, and Cr, according to Tessler et al. is known for its substantial human activity, low GDP, illegal slums (1987), Eysink et al. (1988) and Cetesb (1988, 2000, 2007). The located within estuarine regions, and a lack of wastewater treat- area known as Cananéia (CAN) is located 50 km to the southwest. ment and solid waste collection (Cetesb, 2007; Kirschbaum et al., It includes preserved mangroves and excellent environmental 2009; Cordeiro and Costa, 2010), the latter of which is intensified quality, and uçá crab capture is substantial there (Duarte et al., by 11 industrial sources of pollution and the long-standing illegal 2014). Therefore, this region is important in analyses of the sub- deposition of organochlorine compounds such as lethal impacts on this species (Mendonça and Miranda, 2008;

Table 1 Characteristics of the municipalities/areas sampled in the state of São Paulo (Brazil).

i Characteristic of Central Coast Southern Coast Municipality/Area Cubatão São Vicente Bertioga Mean7SD Total ∑ Iguape Cananéia Juréia Mean7SD Total ∑ ....Dat ta./EooiooyadEvrnetlSft 3 21)176 (2016) 133 Safety Environmental and Ecotoxicology / al. et Duarte L.F.d.A.

Main economic Industry Commerce and Tourism and Tourism, ﬁsh- Fishing Ecotourism and activity services commerce ing and sustainable commerce extraction

1 (km) 142,879 147,893 490,148 – 780,920 1,977,957 1,239,376 7924 – 3,225,257 Área Number of 118,72 332,445 47,645 – 391,962 28,841 12,226 1285* – 42,352 inhabitants 1 Demographic 83,091 224,788 9721 105,8667109 317,600 1458 986 162 8687655 2606 1 2 density (hab./km ) HDI1 0.737 0.768 0.730 0.74570.20 – 0.726 0.720 – 0.72370.04 – GDP2 ( R$ 1000.00) 63,481.45 38,358.00 8855.89 36,898727,342 110,695.34 324.77 1347.06 – 835.917722.86 1671.83 GDP per capita 2 (R$) 52,772.36 11,388.65 17,604.74 27,255722,315 81,765.75 11,177.76 11,027.28 – 11,102 710,640 22,205.04 HPI3 (%) 21.30 15.00 25.00 20.4375.05 61.30 33.30 37.30 – 35.30728.28 70.60 Sanitation 4 (%) 75.14 87.08 30.81 64.34729.64 –––––– Drainage basin5 24,394 8581** 61,291 – 94,266 ––– – 17,068 (km2) Metals (water and As 6,Cd6,12 ,Pb6,11,14 ,Cu6,Cr6,11 ,Hg6,11 , Pb6,Cu6,7,13 ,Hg6,11 , As6,Cr6,11 , – 8 Contaminants Pb 9, 12 ,Cu12 Cd10 ––3 Contaminants sediment) Ni6,11 ,Zn6,11 Ni6,7 ,Cr7,11 ,Zn7,Cd12 Ni6,11 ,Cd11 Organochlorine pes- Alpha-BHC6, Delta-BHC 6, Gamma- Alpha-BHC 6, Beta- –– 4 Contaminants – DDT 8, DDE 8, ––4 Contaminants 6 6 6 8 8 ticides (water andBHC BHC , Delta-BHC , DDD , HCB 6 sediment) Gamma-BHC cdscordatus Ucides 6 ––– ––– – – ctxcl nio.Sf (2016), Saf. Environ. Ecotoxicol. . PCBs (Sediment) PCBs totais 1 Contaminant PAHs (Sediment)6 Acenaphthene, Acenaphthylene, An- Acenaphthylene, An- –– 12 Contaminants ––– – – thracene, Benzo-a-Anthracene, Ben- thracene, Benzo-a-

zo-a-Pyrene, Chrysene, Dibenzo-a- Anthracene – Anthracene, Phenanthrene, Fluor- 187 anthene, Fluorene, Naphthalene, Pyrene Lnau,16)(Brachyura, 1763) (Linnaeus,

HDI: Human Development Index, GDP: Gross Domestic Product, GDP per capita: GDP/number of inhabitants, HPI: Human Poverty Index, PCB: Polychlorinated Biphenyl and PAH: Polycyclic Aromatic Hydrocarbon. References in 1 2 3 4 5 ﬁ 6 Table 2: IBGE BRASIL (2010); IBGE BRASIL (2012); IBGE BRASIL (2000);*Nunes (2003), AGEM, Agência Metropolitana da Baixada Santista (2010), CBH Comitês de Bacias Hidrográ cas (2016), **To São Vicente Island, Cetesb 7(2001)Carmo, et al. (2011), 8Reigada et al. (2014), 9Sant’anna et al. (2014), 10 Mahiques et al. (2013), 11 Semensatto-Jr. et al. (2007), 12 Abessa and Ambrozevicius (2008), 13 Mahiques et al. (2009), 14 Cesar et al. (2006) and 15 Pinheiro et al. (2013). The use of a hyphen (-) represents information that is not available. http://dx.doi. 179 180 L.F.d.A. Duarte et al. / Ecotoxicology and Environmental Safety 133 (2016) 176–187

Pinheiro et al., 2012, 2013). The mangroves in Juréia (JUR) are lo- of the previously prepared slides, which were kept in a humidified cated approximately 50 km northeast of Iguape, and this area is in dark chamber (an acrylic box with paper moistened with distilled an ecological station known as the Juréia-Itatins Ecological Station water) for 15 min so that the cells would better adhere to the (EEJI). The human population is more limited in the region and is slides. In addition, 40 μL of working solution was dripped on the based on traditional extractive activities that are free of con- hemocytes on each slide, and these slides were then kept in the taminants (Marques and Duleba, 2004; Pinheiro et al., 2013). The humidified dark chamber for 15 min. Slides were covered using station is therefore considered effective in protecting local biodi- coverslips, and excess liquid was carefully removed using a filter versity (Bruner et al., 2001). In summary, Cananéia and Juréia are paper. In the first hour, the slides were examined every 15 min considered pristine estuarine regions of São Paulo State (Azevedo under an optical microscope (400x) and, if necessary, after the et al., 2009) where some cytological and hematological analyses in second hour, every 30 min. In contrast with micronucleus test, sea bass fish have not shown any sublethal damage (Kirschbaum only granulocytes and semi-granulocytes were evaluated (Hose et al., 2009; Seriani et al., 2013). et al., 1990; Martinez et al., 1999; Matozzo and Marin, 2010). Ly- sosome size and color were determined to assess stress level ab- 2.2. Multi-level biological responses exhibited by U. cordatus normalities, as was cell shape (irregular¼healthy; circular¼ stressed), these characteristics may also be altered by con- 2.2.1. Genotoxicity via micronucleus Test (MN‰): molecular level taminants (Collier et al., 2013). Details of the criteria used to define During the winter of 2013, adult male U. cordatus specimens granulocytes and semi-granulocytes as healthy or under stress are (carapace width or CW 460 mm) were captured by hand (see described by Lowe et al. (1995). Pinheiro and Fiscarelli (2001)). They were in the intermolt stage and free of parasites. Five specimens per subarea were obtained 2.2.3. Condition factor and crab density: population level (n¼15/area), for a total of 90 specimens for the genotoxicity Fulton's condition factor (K) was measured in mature males analysis. The animals collected underwent biometric procedures, from each area. It was calculated using on the following equation: including body size (CW) measurement using precision calipers b (0.01 mm) and total wet weight (WW) measurement using a KWWCWiii= /() precision scale (0.01 g). A hemolymph sample was obtained from where WW is the wet weight of the i-nth individual, recorded each crab in order to conduct the micronucleus test following a i using a digital precision balance (0.01 g), CWi is the carapace modified version of the protocol offered by Scarpato et al. (1990) width of the i-nth individual, measured using precision calipers and successfully employed by Nudi et al. (2010) and Pinheiro et al. (0.05 mm), and b is the constant growth in weight, determined (2013). The number of hemocytes (hyalinocytes) that contained fi micronuclei was recorded in relation to every 1000 cells analyzed using the t of the empirical points of the WW CW relation for ¼ b (MN‰), thus avoiding possible errors if granulocyte and semi- each area based on the power function (WW aCW ), as indicated granulocyte hemocytes were used (Hose et al., 1990; Martinez by Lima-Junior et al. (2002) and Pinheiro and Fiscarelli (2009). et al., 1999; and Matozzo and Marin, 2010). Micronucleated cells Therefore, the mean condition factor was calculated for each were identified according to the descriptions offered by Country- mangrove area and the differences were analyzed. The U. cordatus man and Heddle (1976), and were confirmed by the presence of density of each mangrove subarea was estimated using the pro- cytoplasmic corpuscles less than 1/3 of size of the nucleus (which tocol established by Pinheiro and Almeida (2015). In this case, the 2 were not connected to the micronuclei and which needed to ex- areas were comprised of four squares of 5 5 m (25 m ) to which hibit similar staining results). Cells with more than three micro- random sampling was applied. Two sites were near the margins of nuclei were not considered. the estuary (approximately 15 m away from the estuary water) with 50 m between them, and the other two were positioned 2.2.2. Cytotoxicity according to neutral red retention time (NRRT): more deeply inside the forest (about 30 m away from the banks of 2 physiological level the estuary). The total was therefore 100 m per subarea. The 2 Hemolymph samples were obtained from 10 specimens per density (ind m ) of each sample square was determined using subarea (n¼30/area), for a total of 180 samples for the cytotoxicity the indirect method based on the number of open and closed assay. First, the NRRT assay was applied to U. cordatus, with an burrows with biogenic activities (Pinheiro and Almeida, 2015)in adaptation to the protocol originally created by Lowe et al. (1995) which the presence of one crab per burrow is assumed (Wun- for bivalve mollusks and subsequently adapted to crabs (Buratti derlich et al., 2008). U. cordatus density was represented as the et al., 2012). The slides were decontaminated with nitric acid (5%) average obtained in each mangrove area from twelve sampling 2 and then washed, air dried, and treated with a diluted poly-L-ly- units (300 m ). sine solution (1:10 in distilled water) using 10 μL per slide for hemocyte adhesion. The four following solutions were prepared: 2.3. Environmental quality indicator: quantity of solid waste (1) a physiological solution (4.77 g of HEPES, 25.48 g of sodium chloride, 4.36 g of magnesium sulfate, 0.75 g of potassium chlor- In addition to the environmental impacts obtained from a re- ide, 1.92 g of calcium chloride, 1 L of distilled water, and pH 7.36); view of the literature, the quantity of solid waste (g m 2)by (2) a stock neutral red solution (22.8 mg of neutral red and 1 mL of mangrove area or subarea was also used as an indicator of en- DMSO); (3) a working solution (10 μL of the stock solution and vironmental quality. Solid waste is also a source of contamination 5 mL of physiological solution); and (4) an anticoagulant solution for U. cordatus, and it was assumed that many types of con- (2.05 g of glucose, 0.8 g of sodium citrate, 0.42 g of sodium chlor- taminants are higher in places where there is a greater human ide, and 100 mL of distilled water). The anticoagulant and phy- presence (Abessa and Ambrozevicius, 2008; Cordeiro and Costa, siological solutions were mixed (a 3:2 ratio) using a 0.5-mL filled 2010). To achieve this information, waste found in the sediment hypodermic syringe (1 mL, provided with a 21 G needle). These was collected from the same square sample used to estimate crab syringes were used to extract 0.5 mL of hemolymph per crab, and density. These items were placed in plastic bags and transported to the samples were transferred into microtubes (2 mL) that were the laboratory, where they were classified according to type slightly homogenized and then placed onto specific shelves for 15– (processed wood, plastic, glass, etc.), washed to remove mud, and 20 min. Next, 40 μL of a hemolymph, anticoagulant and physio- dried in a forced ventilation oven (60 °C for 72 h), until a constant logical solution were dripped from each microtube on the surface weight was achieved on a digital precision scale (0.01 g). Dry

2 Fig. 2. Solid waste volume (g m ) as an environmental impact indicator observed 4. Discussion in mangrove areas in the state of São Paulo, Brazil. Bars indicate the average volume, vertical lines indicate standard deviation, and different letters above the bars represent statistical differences between mangrove areas as per ANOVA and Tu- The use of biomarker responses such as micronuclei and lyso- key's test (Po0.05). somal integrity in different species inhabiting various sites across

10 c A Conservation Hierarchy 9 Indicator of Mangrove Areas 8 b 7 b b Genotoxicity (MN‰) CUB > (BET = SAV) ≥ IGU > CAN > JUR 6 Citotoxicity (NRRT) SAV > (CUB = BET = IGU) > (JUR = CAN) 5 4 Crab Density (DENS) (BET = CUB = SAV) ≤ IGU ≤ (CAN = JUR)

MN/1,000 cells 3 Solid Waste (SW) SAV ≥ (CUB = [BET = IGU]) 2 a a 1 Chart 1. Decreasing hierarchy of mangrove area conservation based on average values for each indicator and according to their statistical differences (ANOVA; 0 Po0.05). CUB¼Cubatão; BET¼Bertioga; SAV¼São Vicente; IGU¼Iguape; Bertioga Cubatão São Vicente Iguape Cananéia Juréia CAN¼Cananéia; and JUR¼Juréia. 350 B e the region covered by the OSPAR Convention revealed the re- 300 levance of cytotoxicity and genotoxicity levels in ecosystem as- 250 sessments. Lysosomal membrane stability has recently been adopted by the UNEP as part of the ﬁrst tier of techniques for 200 d assessing harmful impacts within the Mediterranean Pollution 150 Programme (Ospar, 2013). NRRT (min.) The rationale for applying cytogenetic assays to ecologically 100 c b bc relevant species is that, after the contaminants reach cells, whe- 50 a ther by contact, absorption, or ingestion (Rainbow, 1997, 2007;

0 Rainbow and Black, 2005; Ahearn et al., 2004), concentrations that Bertioga Cubatão São Vicente Iguape Cananéia Juréia exceed animals’ physiological capacities (metabolism/excretion) may be immobilized by detoxification processes, thus avoiding 3 b C sublethal damage (Tsarpali and Dailianis, 2012; Newman, 2013). b 2,5 Contaminants may also reach generic common targets (e.g. lyso- a ab somes or nuclei), a process which damages genetic material and ) 2 causes dysfunction of cellular processes. This process has been a a mechanistically linked with many aspects of pathology associated 1,5 with toxicity and degenerative diseases (Moore et al., 2006). Ge- netic damage can lead to genetic erosion and may especially 1 Density (ind.m Density compromise species resilience to selective pressures in the environment (Bijlsma and Loeschcke, 2012). Early physiological da- 0,5 mage includes lipid peroxidation of the cell membranes caused by free radicals that alter their structures and permeability, a change 0 Bertioga Cubatão São Vicente Iguape Cananéia Juréia which may result in apoptosis. This process is associated with natural aging and may occur naturally in organisms (Tsarpali and Dailianis, 2012; Newman, 2013). However, sublethal changes are potentiated by xenobiotic activity (Wei and Yang, 2015). Fig. 3. Biological responses of the mangrove crab Ucides cordatus by mangrove area This study presents baseline values, which may be understood in state of São Paulo (Brazil), according to: (A) the number of micronucleated cells as normality parameters (or references) for determining cytotox- per thousand (MN‰); (B) neutral red retention time assay (NRRT), in minutes; and (C) crab density (ind m2). Bars indicate the average, vertical lines indicate the icology and genotoxicity in studies on U. cordatus. Scarpato et al. standard deviation, and different letters above the bars reflect statistical differences (1990) defined normal genotoxicity values as MN‰o4, but this between averages according to ANOVA and a post hoc Tukey test (Po0.05). threshold is based on the responses of bivalve mollusk specimens (Mytilus galloprovincialis) unaffected by xenobiotics. Therefore, 300 interspecific variation is expected. Fossi et al. (2000) confirmed y = 153.58x-0.424 this value for the crab Carcinus aestuarii, but according to Nudi R² = 0.61 et al. (2010), U. cordatus exhibits greater genetic sensitivity. This argument was confirmed in the current study; areas with lower 200 human impact (Cananéia) and pristine areas (Juréia) had lower genotoxicity averages (MN‰o2). Therefore, it is possible to as- sume this value as a mean genotoxicity baseline for U. cordatus, 100 thus allowing for the categorization of specimens from more im- NRRT (min.)NRRT pacted mangrove areas such as Cubatão and Bertioga. Meanwhile, São Vicente genotoxicity values were found to be four times higher than this reference value. In a study consistent with this evidence, 0 Pinheiro and Toledo (2010) reported a genotoxicity value of 11.5 0246810 MM‰ in one specimen with cheliped malformation captured in MN/1,000 cells São Vicente, which is five times higher than the environmental quality baseline established in this study. Fig. 4. Relationship between NRRT (time of lysosome integrity by neutral red assay, in minutes) and MN‰ (number of micronucleated cells per thousand analyzed) in In the case of cytotoxicity, the baseline substantially surpassed the mangrove crab Ucides cordatus, with fit of the empirical points via power the values reported in other studies on species from different function. taxonomic groups (Svendsen et al., 2004; Buratti et al., 2012). The

Fig. 5. Principal Component Analysis (PCA) using the biological parameters (MN‰¼frequency of micronucleated cells per thousand; NRRT¼time of integrity of lysosomes by neutral red assay; K¼condition factor; and DENS¼crab density), based on the crab Ucides cordatus, and its relationship with environmental quality indicator in mangroves (SW, solid waste volume). A¼a description of both dimensions (DIM 1 and DIM 2); B¼dendrogram of the PCA and the three groups generated; and C¼distances of similarity among the six mangrove areas. The square icons in different colors represent the averages of biological parameters and solid waste volume of each mangrove area along São Paulo's central coast (BET¼Bertioga; CUB¼Cubatão; SAV¼São Vicente) and southern coast (CAN¼Cananéia; IGU¼Iguape; and JUR¼Juréia). average NRRT for the crab Carcinus maenas was 70 min in the powerful in environmental monitoring studies (Adams, 1990; control treatment (Buratti et al., 2012; Aguirre-Martínez et al., Fishelson et al., 1999). Crab density is a population parameter for 2013). The NRRT was almost three times higher (205 min) when which establishing a baseline (reference) is more complex (Pauly, the same nature experiments were applied to the shrimp Fenner- 1995), since environmental support capacity (ecosystem function) openaeus chinensis (Yao et al., 2008). In the Juréia mangroves, the is the most important factor in determining population size average NRRT for U. cordatus specimens was 260 min (742.7), (Gamfeldt et al., 2015). As an example, mangroves of the northern with a reduced coefficient of variation (13.3%). This result reflects and northeast regions of Brazil hold more biomass than those in the reliability of the estimate, as well as the pristine condition of the south and southeast (Schaeffer-Novelli and Cintrón, 1986). This this ecological station. During the experiment, only cells of crabs difference is reflected in differences in U. cordatus density, which is from Juréia and Cananéia displayed dye leakage from the lyso- higher at more productive sites; it ranges from 3.7 ind m2 (Goes somes into the cytosol approximately two hours after exposure. et al., 2010) to 5.7 ind m2 (Fernandes and Carvalho, 2007). In The effects of contamination on the parameters involving po- contrast, less productive mangrove areas such as those in south- pulation dynamics are also difficult to correlate, though Jergentza eastern and southern Brazil may exhibit densities that are three to et al. (2004) and Brink et al. (2007) have linked this negative in- five times lower. Examples of densities reported in these regions fluence to other crustacean species. Population indicators usually include 1.1 ind m2 (Branco, 1993), 1.7 ind m2 (Blankensteyn respond belatedly to the chronic effects of disturbances to the et al., 1997), and 2.1 ind m2 (Wunderlich et al., 2008). However, natural environment and are therefore less sensitive and less the sampling in the current study was standardized in similar

2000; Neri et al., 2003; Svendsen et al., 2004; Terlizzi et al., 2005). Table 2 Because of this, sublethal damages may be considered to be pre- Damage categories suggested for mangrove areas (PNI¼probably no impact, PLI¼ pathological indicators (Tsarpali and Dailianis, 2012), despite a lack probable low impact, and PHI¼probable high impact), with an indication of of findings showing an impact on the population. This study baseline values for genotoxicity (MN‰), cytotoxicity (NRRT), and crab density (DENS) for U. cordatus, based on solid waste volume and historic contamination of confirms the association between sublethal damage and popula- the mangrove areas in state of São Paulo, Brazil: Central Coast (BET¼Bertioga, fi tion parameters (crab density), a nding which supports the eco- CUB¼Cubatão, and SAV¼São Vicente) and Southern Coast (CAN¼Cananéia, logical effectiveness of the biomarkers. The results also show that, JUR¼Juréia, and IGU¼Iguape). the crabs experienced more sublethal damage and lower population densities in the more highly impacted areas (Cubatão and Damage Genotoxicity Cytotoxicity Crab den- Mangrove category (MN/1000) (NRRT, in min.) sity (ind. areas Bertioga). The opposite was found to occur in the areas of least m2) impact (Juréia and Cananéia) (see Tables 1 and 2). These results indicate that, in the case of U. cordatus, the cellular–level para- PNI o3 4120 41.7 JUR and CAN meters (MN‰ and NRRT) combined with population-level para- PLI 3–560–120 1–1.7 IGU PHI 45 o60 o1 CUB, SAV and meters (density) are sufficiently effective in diagnosing the quality BET of the mangroves studied using a multi-level biological responses

Please cite this article as: Duarte, L.F.d.A., et al., Multi-level biological responses in Ucides cordatus (Linnaeus, 1763) (Brachyura, Ucididae) as indicators of conservation status in mangrove areas from the western atlantic. Ecotoxicol. Environ. Saf. (2016), http://dx.doi. org/10.1016/j.ecoenv.2016.07.018i L.F.d.A. Duarte et al. / Ecotoxicology and Environmental Safety 133 (2016) 176–187 185 can direct financial resources to optimal studies on xenobiotics in pesqueiros e conteúdo protéico do caranguejo do mangue Ucides cordatus impacted mangrove areas. Only with a more specific approach and (Ocypodidae) nos manguezais da Baía das Laranjeiras Pr. Arq. Biol. Tecnol. 40 – fi (2), 331 349. a more re ned sampling grid are we able to identify the main Bonassi, S., Hagmar, L., Strombert, U., Montagud, A.H., Tinnerbert, H., Forni, A., sources of pollution in order to ultimately reduce it and mitigate Heikkila, P., Wanders, S., Wilhardt, P., Hansteen, I.L., Knudsen, L.E., Norppa, H., its effects. 2000. European study group on cytogenetic biomarkers and heath chromoso- mal aberrations in lymphocytes predict human cancer independently of exposure to carcinogens. Cancer Res. 60, 1619–1625. Branco, J.O., 1993. Aspectos bioecológicos do caranguejo Ucides cordatus (Linnaeus Acknowledgments 1763) (Crustacea, Decapoda) do manguezal do Itacorubi, Santa Catarina, BR. Arq. Biol. Tecnol. 36 (1), 133–148. Brasil, 2006. Departamento de Água e Energia Elétrica. Plano Estadual de Recursos The authors would like to thank the São Paulo State Research Hídricos: 2004/2007 – Resumo. São Paulo: DAEE e Conselho Estadual de Re- Foundation (FAPESP) for awarding financial aid to MAAP for the cursos Hídricos. 92p. Uçá III Project (FAPESP Case # 2009/14725-1) and a Ph.D. scho- Brink, P.J.V., Baveco, J.M.H., Verboom, J., Heimbach, F., 2007. An individual-based approach to model spatial population dynamics of invertebrates in aquatic larship to LFAM (Case # 2010/01552-9). The authors are also ecosystems after pesticide contamination. Hazard/Risk Assess. 26 (10), grateful to the Brazilian National Council for Scientific and Tech- 2226–2236. fi Bruner, A.G., Gullison, R.E., Rice, R.E., Fonseca, G.A.B., 2001. Effectiveness of parks in nological Development (CNPq) for a scienti c productivity grant – – protecting tropical biodiversity. Science 291, 125 128. (triennium 2010 2012) to MAAP (Case # 302813/2010-1) and Brunetti, R., Majone, F., Gola, I., Beltrame, C., 1988. The micronucleus test: examples CDSP. The authors would like to thank biologists Pablo P.G. Silva, of application to marine ecology. Mar. Ecol. Prog. Ser. 44, 65–68. Sérgio O. Asche, Vanessa S. Soares, Bruna T. Souza, Kátia V.C. Sousa, Bijlsma, R., Loeschcke, V., 2012. Genetic erosion impedes adaptative responses to stressful environments. Evolut. Appl. 5 (2), 117–129. Stephany C. Leitão, Fernanda F. Lopes, Gilmar Giraldelli, Leonardo Buratti, S., Ramos-Gómez, J., Fabbrini, E., Delvalls, T.A., 2012. Application of neutral M. S. Mesquita, Luis F. Natálio, Maria E. Laranjeira, Michel T. An- red retention assay to caged clams (Ruditapes decussatus) (and crabs (Carcinus geloni, Rodolfo Probst, Rodrigo F. Malimpensa and André L.P. Souza maena)s) (in the assessment of dredged material). Ecotoxicology 21, 75–86. fi Burgeot, T., His, E., Galgani, F., 1995. The micronucleus assay in Crassostrea gigas for for their support in the eld expeditions and laboratory activities. the detection of seawater genotoxicity. Mutat. Res. 342, 125–140. In additions, the authors are grateful to Gustavo H.S. Pinheiro for Cannicci, S., Bartolini, F., Dahdouh-Guebas, F., Fratini, S., Litulo, C., Macia, A., Mrabu, preparing the map and icons representing the mangrove areas in E.J., Penha-Lopes, G., Paula, J., 2009. Effects of urban wastewater on crab and mollusc assemblages in equatorial and subtropical mangroves of East Africa. this study. Data was obtained with permanent authorization to Estuar. Coast. Shelf Sci. 84, 305–317. collect zoological material provided by the Brazilian Institute of Caricato, R., Lionetto, M.G., Schettino, T., 2010. Seasonal variation of biomarkers in the Environment and Renewable Natural Resources (SISBIO/IBA- Mytilus galloprovincialis sampled inside and outside Mar Piccolo of Taranto – MA) to MAAP (# 13581-1), as well as the authorization from the (Italy). Chem. Ecol. 26 (1), 143 153. Carmo, C.A., Abessa, D.M., Neto, J.G.M., 2011. Metais em águas, sedimentos e peixes Brazilian Forest Institute's Scientific-Technical Commission (CO- coletados no estuário de São Vicente -SP, Brasil. O Mundo da Saúde 35 (1), TEC/FF) (SMA Case # 260108-009.809/2010), to perform sample 64–70. collections in the Juréia-Itatins Ecological Station (JIES). Cavas, T., Garanko, N.N., Victor, V. Arkhipchuk, 2005. Induction of micronuclei and binuclei in blood, gill and liver cells of fishes subchronically exposed to cadmium chloride and copper sulphate. Food Chem. Toxicol. 43 (4), 569–574. Cbh, 2016. Comitê de Bacias Hidrográficas. Sistema integrado de gerenciamento de References recursos hídricos do estado de São Paulo. Disponível em: 〈http://www.sigrh.sp. gov.br〉, (acessed 27.01.16). Cesar, A., Pereira, C.D.S., Santos, A.R., Abessa, D.M.S., Fernández, N., Choueri, R.B., Abessa, D.M.S., Ambrozevicius, A.P., 2008. Poluição aquática e tratamento de es- Delvalls, T.A., 2006. Ecotoxicological assessment of sediments from the Sanos gotos, Cap. 04, 41–54p. In: Oliveira, A.J.F.C., Pinheiro, M.A.A., Fontes, R.F.C. and São Vicente Estuarine System - Brazil. Braz. J. Oceanogr. 54 (1), 55–63. (Orgs.). Panorama Ambiental da Baixada Santista. Universidade Estadual Pau- Cetesb, 1988. Companhia de Tecnologia de Saneamento Ambiental. Metais pesados lista - Campus Experimental do Litoral Paulista, 1ª Edição, ISBN 978-85-61498- no Vale do Ribeira e em Iguape - Cananéia. São Paulo. 7p. 02-3, São Vicente (SP), 127p. Cetesb, 2000. Companhia de Tecnologia de Saneamento Ambiental. Avaliação da Adam, M.L., Torres, R.A., Sponchiado, G., Motta, T.S., Oliveira, C.M.R., Carvalho-Filho, qualidade do Rio Ribeira de Iguape e afluentes, São Paulo, 33p. M.A., Correia, M.T.S., 2010. Environmental degradation at a public park in Cetesb, 2001. Companhia de Tecnologia de Saneamento Ambiental. Sistema Es- Southern Brazil as revealed through a genotoxicity test (MN) on peripheral tuarino de Santos e São Vicente. Relatório do Programa de Controle de Poluição. blood cells from Poecilia vivipara (Teleostei). Water Air Soil Pollut. 211, 61–68. São Paulo (SP), 137p þ46 pranchas. Adams, S.M., 1990. Status and use of biological indicators for evaluating the effects Cetesb, 2007. Companhia de Tecnologia de Saneamento Ambiental. Relação de of stress on fish. Am. Fish. Soc. Symp. 8, 1–8. áreas contaminadas - Novembro de 2007 - Relação por municípios. 〈http:// Agem, 2010. Agência Metropolitana da Baixada Santista. Disponível em: 〈http:// www.cetesb.sp.gov.br/Solo/areas_contaminadas/texto_areas_cont_nov_08.pdf〉, www.agem.sp.gov.br〉, (acessed 27.01.16). (acessed 02.08.15). Aguirre-Martínez, G.V., Buratti, S., Fabbri, E., Del Valls, T.A., Martín-Díaz, M.L., 2013. Christofoletti, R.A., Hattori, G.Y., Pinheiro, M.A.A., 2013. Food selection by a man- Environ. Monit. Assess. 185, 3783–3793. grove crab: temporal changes in fasted animals. Hydrobiologia 702, 63–72. Ahearn, G.A., Manadal, P.K., Mandal, A., 2004. Mechanisms of heavy-metal se- http://dx.doi.org/10.1007/s10750-012-1307-6. questration and detoxification in crustaceans: a review. J. Comp. Physiol. B 174, Collier, T.K., Chiang, M.W.L., Au, D.W.T., Rainbow, P.S., 2013. Biomarkers currently 439–452. used in environmental monitoring. In: Amiard-Triquet, C., Amiard, J.C., Rain- Amiard-Triquet, C., Cossu-Leguille, C., Mouneyrac, C., 2013. Biomarkers of defense, bow, P.S. (Eds.), Ecological Biomarkers. CRC Press/Taylor & Francis Group, Uni- tolerance and ecological consequences. In: Amiard-triquet, C., Amiard, J.C., ted States, p. 450. Rainbow, P.S. (Eds.), Ecological Biomarkers. CRC Press/Taylor & Francis Group, Cordeiro, C.A.M.M., Costa, T.M., 2010. Evaluation of solid residues removed from a United States, p. 450. mangrove swamp in the São Vicente Estuary, SP, Brazil. Mar. Pollut. Bull. 60, Arslan, O.C., Boyacioğlu, M., Parlak, H., Katalay, S., Karaaslan, M.A., 2015. Assessment 1762–1767. of micronuclei induction in peripheral blood and gill cells of some fish species Countryman, P.I., Heddle, J.A., 1976. The production of micronuclei from chromo- from Aliağa Bay Turkey. Mar. Pollut. Bull. 94 (1–2), 48–54. some aberrations in irradiated cultures of human lymphocytes. Mutat. Res. 41, Azevedo, J.S., Serafim, A., Braga, E.S., Fávaro, D.I., Bebianno, M.J., 2009. Biomarkers of 321–332. exposure to metal contamination and lipid peroxidation in the benthic fish Cunha-Lignon, M., 2014. Monitoramento de bosques de mangue do Estado de São Cathorops spixii from two estuaries in South America, Brazil. Ecotoxicology 18 Paulo, Brasil. Relatório Final de Pesquisa, CNPq Edital Universal (Proc. no. (8), 1001–1010. 472419/2011-0), Período 01/2012 - 01/2014, 52p. Barrilli, G.H.C., Rocha, O., Negreiros, N.F., Verani, J.R., 2015. Influence of environ- Cunha-Lignon, M., Kampel, M., Dahdouh-Guebas, F. 2011a. Urban mangrove dy- mental quality of the tributaries of the Monjolinho River on the relative con- namics under increasing anthropogenic pressure: Analysis of remote sensing dition factor (Kn) of the local ichthyofauna. Biota Neotrop. 15 (1), 1–9. data and Brazilian legislation. In: Proceedings of the International Conference: Beltrame, M.O., De Marco, S.G., Marcovecchio, J.E., 2010. Influences of sex, habitat, Meeting on Mangrove Ecology, Functioning and Management (MMM3), VLIZ and seasonality on heavy-metal concentrations in the burrowing crab (Neo- Special Publication 57, 47. helice granulata) from a coastal lagoon in Argentina. Arch. Environ. Contam. Cunha-Lignon, M., Kampel, M., Menghini, R.P., Schaeffer-Novelli, Y., Cintrón, G., Toxicol. 58 (3), 746–756. Dahdouh-Guebas, F., 2011b. Mangrove forests submitted to depositional pro- Beltrame, M.O., De Marco, S.G., Marcovecchio, J.E., 2011. The burrowing crab Neo- cesses and salinity variation investigated using satellite images and vegetation helice granulata as potential bioindicator of heavy metals in estuarine systems structure surveys. J. Coast. Res. 64 (1), 344–348. of the Atlantic coast of Argentina. Environ. Monit. Assess. 172, 379–389. Dailianis, S., Domouhtsidou, G.P., Raftopoulou, E., Kaloyianni, M., Dimitriadis, V.K., Blankensteyn, A., Cunha Filho, D., Freire, A.S., 1997. Distribuição, estoques 2003. Evaluation of neutral red retention assay, micronucleus test,

acetylcholinesterase activity and a signal transduction molecule (cAMP) in and haemocytes of fresh water snails exposed to mercuric chloride. Int. J. Re- tissues of Mytilus galloprovincialis (L.), in pollution monitoring. Mar. Environ. cent Sci. Res. 6 (8), 5725–5730. Res. 56, 443–470. Kaufman, L., Rousseeuw, P.J., 1990. Finding groups in data: an introduction to Deguchia, Y., Toyoizumi, T., Masuda, S., Yasuhara, A., Mohri, S., Yamada, M., Inoue, Y., cluster analysis. Wiley, New York, 283p. Kinae, N., 2007. Evaluation of mutagenic activities of leachates in landfill sites Kirschbaum, A.A., Seriani, R., Pereira, C.D., Assunção, A., Abessa, D.M.D.S., Rotundo, by micronucleus test and comet assay using goldfish. Mutat. Res./Genet. Tox- M.M., Ranzani-Paiva, M.J., 2009. Cytogenotoxicity biomarkers in fat snook icol. Environ. Mutagen. 627 (2), 178–185. Centropomus parallelus from Cananéia and São Vicente estuaries, SP, Brazil. Depledge, M.H, Amaral-Mendes, J.J., Daniel, B., Halbrook, R.S., Kloepper-Sams, P., Genet. Mol. Biol. 32 (1), 151–154. Moore, M.N., Peakall, D.B. 1993. The Conceptual Basis of the Biomarker Ap- Klobučar, G.I.G., Malev, O., Šrut, M., Štambuk, A., Lorenzon, S., Cvetković, Z., Ferrero, proach. In: Peakall, D.B., Shugart, L.R. (Eds.) Biomarkers. NATO ASI Series, 119p. E.A., Maguire, I., 2012. Genotoxicity monitoring of freshwater environments Dissanayake, A., Galloway, T.S., Jones, M.B., 2011. Seasonal differences in the phy- using caged crayfish (Astacus leptodactylus). Chemosphere 87 (1), 62–67. siology of Carcinus maenas (Crustacea: Decapoda) from estuaries with varying Krebs, C.T., Burns, K., 1977. Long-term effects of an oil spill on populations of the levels of anthropogenic contamination. Estuar. Coast. Shelf Sci. 93 (4), 320–327. salt-marsh crab Uca pugnax. Science 197 (4302), 484–487. Duarte, L.F.A., Duran, R.S., Mendonça, J.T., Pinheiro, M.A.A., 2014. The fishery for the Krebs, C.T., Valiela, I., 1974. Long-term effects of an oil spill on populations of the ‘uçá’- crab Ucides cordatus (Linnaeus, 1763) in a mangrove area in Cananéia, salt-marsh crab Uca pugnax. Mar. Pollut. Bull. 5 (9), 140–142. State of São Paulo, Brazil. I. Fishery performance, exploitation patterns and Lacher, T.E., Goldstein, M.I., 1997. Tropical ecotoxicology: status and needs. Environ. factors affecting the catches. Braz. J. Oceanogr. 62 (3), 187–199. Toxicol. Chem. 16 (1), 100–111. Eichler, P.P.B., Eichler, B.B., David, C.J., Miranda, L.B., Sousa, E.C.P.M., 2006. The es- Lima-Junior, S.E., Cardone, I.B., Goitein, R., 2002. Determination of a method for tuary ecosystem of Bertioga, São Paulo, Brazil. J. Coast. Res. (Spec. Issue 39), calculation of allometric condition factor of fish. Acta Sci. 24, 397–400. 1110–1113. Lowe, D.M., Valentino, U.F., Depledge, M.H., 1995. Contaminant-induced lysosomal Environment Canada, 1999a. Canadian Sediment Quality Guidelines for the Pro- membrane damage in blood cells of mussels Mytilus galloprovincialis from the tection of Aquatic Life. Summary Tables. 〈http://www.ec.gc.ca〉. Venice Lagoon: an in vitro study. Marine Ecology Progress Series 129, 189–196. Environment Canada, 1999b. Canadian Water Quality Guidelines for the Protection Mahiques, M.M., Burone, L., Figueira, R.C.L., Lavenérer-Wanderley, A.A.O., Capellari, of Aquatic Life. Summary Tables. 〈http://www.ec.gc.ca〉. B., Rogacheski, C.E., Barroso, C.P., Santos, L.A.S., Cordero, L.M., Cussioli, M.C., Eskandari, S., Mozdarani, H., Moradi, A.M., Shahhosseiny, M.H., 2012. Cytogenetic 2009. Anhropogenic influences in a lagoonal environment: a multiproxy ap- damage induced by crude oil in Anodonta cygnea (Mollusca, Bivalvia) assessed proach at the Vaelo Grande Mouth, Cananéia-Iguape System (SE BRAZIL). Braz. by the comet assay and micronucleus test. J. Mar. Sci. Eng. 2 (4), 215–224. J. Oceanogr. 57 (4), 325–337. Eysink, G.G.J., Pádua, H.B., Piva-Bertoletti, A.E., Martins, M.C., Pereira, D.N., 1988. Mahiques, M., Figueira, R., Salaroli, A., Alves, D., Gonçalves, C., 2013. 150 years of Metais pesados no Vale do Ribeira e Iguape-Cananéia. Ambiente: Rev. CETESB anthropogenic metal input in a biosphere reserve: the case study of the Ca- Tecnol. 2 (1), 6–13. nanéia–Iguape coastal system, southeastern Brazil. Environ. Earth Sci. 4 (68), Faraway, J.J., 2002. Practical regression and Anova using R, 212p. Available from 1073–1087. 〈http://cran.r-project.org/doc/contrib/Faraway-PRA.pdf〉. Marques, J.P., Guimarães, C.R., Boas, A.V., Carnaúba, P.U., Moraes, J., 2012. Con- FDEP, 1994. Approach to the assessment of sediment quality in Florida coastal tamination of public parks and squares from Guarulhos (São Paulo State, Brazil) waters. Vol. 1 Development and evaluation of sediment quality assessment by Toxocara spp. and Ancylostoma spp. Rev. Inst. Med. Trop. São Paulo 54 (5), guidelines. Prepared for Florida Department of Environmental Protection, Of- 267–271. fice of Water Policy, Tallahasee, FL, by MacDonald Environmental Sciences Ltd., Marques, O.A.V., Duleba, W., 2004. Estação Ecológica Juréia-Itatins - Ambiente Fí- Ladysmith, British Columbia. sico, Flora e Fauna. Holos, Editora Ltda-ME, Ribeirão Preto, SP, 384p. Fernandes, M.E.B., Carvalho, M.L., 2007. Bioecologia de Ucides cordatus Linnaeus, Martinez, C.B.R., Alvares, E.P., Harris, R.R., Santos, M.C.F., 1999. A morphological 1763 (Decapoda: Brachyura) na costa do Estado do Amapá. Bol. Lab. Hidrobiol. study on posterior gills of the mangrove crab Ucides cordatus. Tissue Cell 31 (3), 20, 14–21. 380–389. Fiscarelli, A.G., Pinheiro, M.A.A., 2002. Perfil sócio-econômico e conhecimento et- Martins, C.C., Bícego, M.C., Mahiques, M.M., Figueira, R.C., Tessler, M.G., Montone, R. nobiológico do catador do caranguejo-uçá, Ucides cordatus (Linnaeus, 1763), nos C., 2011. Polycyclic aromatic hydrocarbons (PAHs) in a large South American manguezais de Iguape (24°41′S), SP, Brasil. Actual. Biol. 24 (77), 129–142. industrial coastal area (Santos Estuary, Southeastern Brazil): sources and de- Fishelson, L., Bresler, V., Manelis, R., Zuk-Rimon, Z., Dotan, A., Hornung, H., Yawetz, positional history. Mar. Pollut. Bull. 63 (5), 452–458. A., 1999. Toxicological aspects associated with the ecology of Donax trunculus Matozzo, V., Marin, M.G., 2010. First cytochemical study of haemocytes from the (Bivalvia, Mollusca) in a polluted environment. Sci. Total Environ. 226, 121–131. crab Carcinus aestuarii (Crustacea, Decapoda). Eur. J. Histochem. 54, 44–49. Fossi, M.C., Casini, S., Savelli, C., Corbelli, C., Franchi, E., Mattei, N., Sanchez-Her- Mendonça, J.T., Miranda, L.V. de, 2008. Estatística pesqueira do litoral sul do estado nandez, J.C., Corsi, I., Bamber, S., Depledge, H.M., 2000. Biomarker responses at de São Paulo: subsídios para gestão compartilhada. Pan-Am. J. Aquat. Sci. 3 (3), different levels of biological organization in crabs (Carcinus aestuarii) experi- 152–173. mentally exposed to benzo)[a]pyrene. Chemosphere 40, 861–874. Monserrat, J.M., Martínez, P.E., Geracitano, L.A., Amado, L.L., Martins, C.M.G., Pinho, Gamfeldt, L., Lefcheck, J.S., Byrnes, J.E.K., Cardinale, B.J., Duffy, J.M., Griffin, J.N., 2015. G.L.L., Chaves, I.S., Ferreira-Cravo, M., Ventura-Lima, J., Bianchini, A., 2007. Marine biodiversity and ecosystem functioning: what's known and what's Pollution biomarkers in estuarine animals: critical review and new perspec- next? Oikos 124, 252–265. http://dx.doi.org/10.1111/oik.01549. tives. Comp. Biochem. Physiol. 146, 221–234. Goes, P., Branco, J.O., Pinheiro, M.A.A., Barbieri, E., Costa, D., Fernandes, L.L., 2010. Moore, M.N., Allen, J.I., Mcveigh, A., 2006. Environmental prognostics: an integrated Bioecology of the uçá-crab, Ucides Cordatus (Linnaeus, 1763), in Vitória Bay, model supporting lysosomal stress responses as predictive biomarkers of ani- Espírito Santo State, Brazil. Braz. J. Oceanogr. 58 (2), 153–163. mal health status. Mar. Environ. Res. 61, 278–304. Gouveia, S.C.R., 2012. Praias arenosas oceânicas do estado de São Paulo (Brasil): Neri, M., Fucic, A., Knudsen, L.E., Lando, C., Merlo, F., Bonassi, S., 2003. Micronuclei síntese dos conhecimentos sobre morfodinâmica, sedimentologia, transporte frequency in children exposed to environmental mutagens: a review. Mutation costeiro e erosão costeira. Revista do Departamento de Geografia, 307–371. Research 544, 243–254. Hagger, J.A., Lowe, D., Dissanayake, A., Jones, M.B., Galloway, T.S., 2010. The in- Newman, M.C., 2013. Bioaccumation. In: NEWMAN, M.C. (Ed.), Quantitative Eco- ffluence of seasonality on biomarker responses in Mytilus edulis. Ecotoxicology toxicology. CRC Press/Taylor & Francis Group, United States, p. 570. 19 (5), 953–962. Nudi, A.H., Wagener, A.L.R., Francioni, E., Sette, C.B., Sartori, A.V., Scofield, A.L., 2010. Harford, A.J., Hogan, A.C., Jones, D.R., Dam, R.A.V., 2015. Ecotoxicology of highly Biomarkers of PAH exposure in crabs Ucides cordatus: laboratory assay and field treated mine waters: lessons from an Australian Mine. Mine Water Environ. 34 study. Environ. Res. 110, 137–145. (1), 75–86. Nunes, M., 2003. Do passado ao futuro dos moradores tradicionais da Estação Hose, J.E., Martin, G.G., Gerard, A.S., 1990. A decapod hemocyte classification Ecológica Juréia-Itatins/SP. Doctoral dissertation. Universidade de São Paulo. scheme integrating morphology, cytochemistry, and function. Biol. Bull. 178, Ospar Commission, 2013. Background document and technical annexes for biolo- 33–45. gical effects monitoring, Monitoring and Assessment Series, Update 2013, OS- Husson, F., Josse, J., Le, S., Mazet, J., 2012. FactoMineR: Multivariate Exploratory Data PAR Commission: London, U.K. Analysis and Data Mining with R. R package version 1.18. http://CRAN.Rproject. Otomo, P.V., Reinecke, S.A., 2010. Increased cytotoxic and genotoxic tolerance of org/package=FactoMineR. Eisenia fetida (Oligochaeta) to cadmium after long-term exposure. Ecotoxicol- Ibge Brasil, 2000. Instituto Brasileiro de geografia e Estatística. Censo Demográfico ogy 19, 362–368. 2000. Disponível em: 〈http://www.ibge.gov.br〉, (acessed 27.01.16). Pauly, D., 1995. Anecdotes and the shifting baseline syndrome of fisheries. Tree 10, Ibge Brasil, 2010. Instituto Brasileiro de geografia e Estatística. Censo Demográfico 1. 2010. Disponível em: 〈http://www.ibge.gov.br〉, (acessed 27.01.16). Pereira, C.D.S., Abessa, D.M.S., Choueri, R.B., Almagro-Pastor, V., Augusto, C., Mar- Ibge Brasil, 2012. Instituto Brasileiro de geografia e Estatística. Censo Demográfico anho, L.A., Díaz, M., Laura, M., Torres, R.J., Gusso-Choueri, P.K., Almeida, J.E., 2012. Disponível em: 〈http://www.ibge.gov.br〉, (acessed 27.01.16). Cortez, F.S., Mozeto, A.A., Silbiger, H.L.N., Sousa, E.C.P.M., Del Valls, T.A., Bainy, A. Ihaka, R., Gentleman, R., 1996. R: a language for data analysis and graphics. J. C.D., 2014. Ecological relevance of Sentinels’ biomarker responses: a multi-level Comput. Graph. Stat. 5 (3), 299–314. approach. Mar. Environ. Res. 96, 118–126. ISA - Instituto Socioambiental, 1998. Diagnóstico socioambiental do Vale do Ribeira. Pereira, C.D.S., Martín-Díaz, M.L., Catharino, M.G.M., Cesar, A., Choueri, R.B., Tani- João Paulo Capobianco (Coord.). ISA, São Paulo. guchi, S., Abessa, D.M.S., Bícego, M.C., Vasconcellos, M.B.A., Bainy, A.C.D., Sousa, Jergentza, S., Pessacq, P., Mugni, H., Bonetto, C., Schulz, R., 2004. Linking in situ E.C.P.M., Delvalls, T.A., 2012. Chronic contamination assessment integrating bioassays and population dynamics of macroinvertebrates to assess agricultural biomarkers’ responses in transplanted mussels - a seasonal monitoring. En- contamination in streams of the Argentine pampa. Ecotoxicol. Environ. Saf. 59 viron. Toxicol. 27, 257–267. (2), 133–141. Pereira, C.D.S., Martín-Díaz, M.L., Zanette, J., Augusto, C., Choueri, R.B., Abessa, D.M. Kandroo, M., Tripathi, N.K., Sharma, I., 2015. Detection of micronuclei in gill cells S., Catharino, M.G.M., Vasconcellos, M.B.A., Bainy, A.C.D., Sousa, Moreira, E.C.P.,

Del Valls, J.H., 2011. Integrated biomarker responses as environmental status Schaeffer-Novelli, Y., Cintrón, G., 1986. Guia para estudo de áreas de manguezal: descriptors of a coastal zone (São Paulo, Brazil). Ecotoxicol. Environ. Saf. 74, Estrutura, Função e Flora. Caribbean Ecological Research, São Paulo, p. 150. 1257–1264. Semensatto-Jr, D.L., Araújo, G.C.L., Funo, R.H.F., Santa-Cruz, J.O.A.N.A., Dias-Brito, D.I. Peters, E.C., Gassman, N.J., Firman, J.C., Richmond, R.H., Power, E.A., 1997. Ecotox- M.A.S., 2007. Metais e não-metais em sedimentos de um manguezal não-po- icology of tropical marine ecosystems. Environ. Toxicol. Chem. 16 (1), 12–40. luído, Ilha do Cardoso, Cananéia (SP). Rev. Pesqui. Geociênc. 34 (2), 25–31. Pinheiro, M.A.A., Almeida, R. 2015. Monitoramento da densidade e da estrutura Seriani, R., Abessa, D.M.D.S., Pereira, C.D., Kirschbaum, A.A., Assunção, A., Ranzani- populacional do caranguejo-uçá, Ucides cordatus (Linnaeus, 1763) (Brachyura: Paiva, M.J.T., 2013. Influence of seasonality and pollution on the hematological Ucididae), Cap. 10, 122–133p. In: Turra, A., Denadai, M.R. Protocolos para o parameters of the estuarine fish Centropomus parallelus. Braz. J. Oceanogr. 61 Monitoramento de Habitats Bentônicos Costeiros - Rede de Monitoramento de (2), 105–111. Habitats Bentônicos Costeiros - ReBentos. ISBN (e-book): 978-85-98729-25-1. Silva, W.L., Matos, R.H.R., Kristosch, G.C., 2002. Geoquímica e índice de geoacu- São Paulo: Instituto Oceanográfico da Universidade de São Paulo, 258p. mulação de mercúrio em sedimentos de superfície do estuário de Santos - Pinheiro, M.A.A., Fiscarelli, A.G. 2001. Manual de Apoio à Fiscalização do Car- Cubatão (SP). Quim. Nova 25 (5), 753–756. anguejo-Uçá (Ucides cordatus). Instituto Brasileiro do Meio Ambiente (IBAMA)/ Sponchiado, G., Reynaldo, E.M.F.L., Andrade, A.C.B., Vasconcelos, E.C., Adam, M.L., Centro de Pesquisa e Gestão de Recursos Pesqueiros do Litoral Sudeste e Sul Oliveira, C.M.R., 2011. Genotoxic Effects in erythrocytes of Oreochromis niloticus (CEPSUL), (1)ª Edição, ISBN 85-88570-02-5, Itajaí, 43p. exposed to nanograms-per-liter concentration of 17β-Estradiol (E2): an as- Pinheiro, M.A.A., Oliveira, A.J.F.C., Fontes, R.F.C., 2008. Introdução ao panorama sessment using micronucleus test and comet assay. Water Air Soil. Pollut. 218, – ambiental da Baixada Santista, Cap. 01, 1 4. In: Oliveira, A.J.F.C., Pinheiro, M.A. 353–360. A., Fontes, R.F.C. (Orgs.). Panorama ambiental da Baixada Santista. Universidade Sueitt, A.P.E., Yamada-Ferraz, T.M., Oliveira, A.F., Botta, C.M.R., Fadini, P.S., Nasci- Estadual Paulista - Campus Experimental do Litoral Paulista, 1ª Edição, ISBN mento, M.R.L., Faria, B.M., Mozeto, A.A., 2015. Ecotoxicological risks of calcium 978-85-61498-02-3, São Vicente, SP, 127p. nitrate exposure to freshwater tropical organisms: laboratory and field ex- Pinheiro, M.A.A., Silva, P.P.G., Duarte, L.F.A., Almeida, A.A., Zanoto, F.F., 2012. Accu- periments. Ecotoxicol. Environ. Saf. 117, 155–163. mulation of six metals in the mangrove crab Ucides cordatus (Crustacea: Uci- Svendsen, C., Spurgeon, D.J., Hankard, P.K., Weeks, J.M., 2004. A review of lysosomal didae) and its food source, the red mangrove Rhizophora mangle (Angios- membrane stability measured by neutral red retention: is it a workable perma: Rhizophoraceae). Ecotoxicol. Environ. Saf. 81, 114–121. earthworm biomarker? Ecotoxicol. Environ. Saf. 57, 20–29. Pinheiro, M.A.A., Duarte, L.F.A., Toledo, T.R., Adams, M.A., Torres, R.A., 2013. Habitat Terlizzi, A., Scuderi, D., Fraschetti, S., Anderson, M.J., 2005. Quantifying effects of monitoring and genotoxicity in Ucides cordatus (Crustacea: Ucididae), as tools pollution on biodiversity: a case study of highly diverse molluscan assemblages to manage a mangrove reserve in southeastern Brazil. Environ. Monit. Assess. in the Mediterranean. Mar. Biol. 148, 293–305. 185 (10), 8273–8285. http://dx.doi.org/10.1007/s10661-013-3172-9. Tessler, G.M., Suguio, K., Robilotta, P.R., 1987. Teores de alguns elementos traço Pinheiro, M.A.A., Toledo, T.R., 2010. Malformation in the crab Ucides cordatus (Lin- metálicos em sedimentos pelíticos da superfície de fundo da região Lagunar naeus, 1763) (Crustacea, Brachyura, Ocypodidae), in São Vicente (SP), Brazil. Cananéia-Iguape. An. Simpósio sobre Ecossistemas Costa Sul Sudeste Bras. 2, Rev. CEPSUL - Biodivers. Conserv. Mar. 1 (1), 1–5. 255–263. Pinheiro, M.A.A., Fiscarelli, A.G., 2009. Length-weight relationship of the carapace Theodore, L., Dupont, R.R., 2012. Environmental Health and Hazard Risk Assess- and condition factor of the mangrove crab Ucides cordatus (Linnaeus, 1763) ment: Principles and Calculations. ISBN 978-1-43-986887-4. 636p. (Crustacea, Brachyura, Ucididae). Braz. Arch. Biol. Technol. 52 (2), 397–406. Torres, R.J., Cesar, A., Pereira, C.D.S., Choueri, R.B., Abessa, D.M.S., Nascimento, M.R. Rainbow, P.S., 1997. Ecophysiology of trace metal uptake in crustaceans. Estuar. Coast. Shelf Sci. 44, 169–175. L., Fadini, P.S., Mozeto, A.A., 2012. Bioaccumulation of polycyclic aromatic hy- Rainbow, P.S., 2007. Trace metal bioaccumulation: models, metabolic availability drocarbons and mercury in oysters (Crassostrea rhizophorae) from Two Brazilian and toxicity. Environ. Int. 33, 576–582. Estuarine Zones. Int. J. Oceanogr., 8. http://dx.doi.org/10.1155/2012/838320. Rainbow, P.S., BLACK, W.H., 2005. Cadmium, zinc and uptake of calcium by two Toufexia, E., Tsarpali, V., Efthimiou, I., Vidali, M.S., Vlastos, D., Dailianis, S., 2013. fi crabs, Carcinus maenas and eriocheir sinensis. Aquat. Toxicol. 72, 45–65. Environmental and human risk assessment of land ll leachate: an integrated Reigada, C.S., Luchini, L.C., Barbieri, E., 2014. Presença de organoclorados em approach with the use of cytotoxic and genotoxic stress indices in mussel and – amostras de água e ostras Crassostrea sp. do Município de Cananeia-SP. O human cells. J. Hazard. Mater. 260, 593 601. fi Mundo da Saúde 38 (1), 66–74. Tsarpali, V., Dailianis, S., 2012. Investigation of land ll leachate toxic potency: an Rocha, A.J.S., Santos, T.C.A., Gomes, V., Bícego, M.C., Barbosa, A.C.R.A., Passos, M.J.A. integrated approach with the use of stress indices in tissues of mussels. Aquat. C.R., Hasue, F.M., Ngan, P.V., 2012. Assessment of trophic transfer of benzo(a) Toxicol. 124–125, 58–65. pyrene genotoxicity from the post-larval pink shrimp F. brasiliensis to the ju- Viana, L.F., Tondato, K.K., Súarez, Y.R., Lima-Junior, S.E., 2014. Influence of en- venile Florida pompano T. carolinus. Environ. Toxicol. Pharmacol. 34 (3), vironmental integrity on the reproductive biology of Astyanax altiparanae 969–976. Garutti & Britski. 2000 in the Ivinhema river basin Acta Sci. Biol. Sci. 36 (2), Sacchi, A., Mouneyrac, C., Bolognesi, C., Sciutto, A., Roggieri, P., Fusi, M., Beone, G.M., 165–173. http://dx.doi.org/10.4025/actascibiolsci.v36i2.21052. Capri, E., 2013. Biomonitoring study of an estuarine coastal ecosystem, the Wunderlich, A.C., Pinheiro, M.A.A., Rodrigues, A.M.T., 2008. Biologia do caranguejo- Sacca di Goro lagoon, using Ruditapes philippinarum (Mollusca: Bivalvia). En- uçá, Ucides cordatus (Crustacea: Decapoda: Brachyura), na Baía da Babitonga, viron. Pollut. 177, 82–89. Santa Catarina, Brasil. Rev. Bras. de. Zool. 25 (2), 188–198. Sant’anna, B.S., Santos, D.M., Marchi, M.R.R.D., Zara, F.J., Turra, A., 2014. Surface- Wei, K., Yang, J., 2015. Oxidative damage of hepatopancreas induced by pollution sediment and hermit-crab contamination by butyltins in southeastern Atlantic depresses humoral immunity response in the freshwater crayfish Procambarus estuaries after ban of TBT-based antifouling paints. Environ. Sci. Pollut. Res. 21 clarkia. Fish. Shellfish Immunol. 43 (2), 510–519. (10), 6516–6524. Yao, C.L., Wu, C.G., Xiang, J.H., Li, F., Wang, Z.Y., Han, X., 2008. The lysosome and Scarpato, R., Migliore, L., Alfinito-Cognetti, G., Barale, R., 1990. Induction of micro- lysozyme response in Chinese shrimp Fenneropenaeus chinensis to Vibrio an- nucleus in gill tissue of Mytillus galloprovincialis exposed to polluted marine guillarum and laminarin stimulation. J. Exp. Mar. Biol. Ecol. 363, 124–129. waters. Mar. Pollut. Bull. 21 (2), 74–80. Zar, J.H., 1999. Biostatistical Analysis, 4th ed. Prentice Hall, United States, p. 663.